JP4494340B2 - Glutamate transporter GLAST function-deficient mice - Google Patents

Description

  The present invention relates to a GLAST knockout mouse deficient in the function of GLAST, which is a kind of glutamate transporter, and a method for producing the same. The present invention also relates to the use of the knockout mouse as a normal-tension glaucoma model mouse and a method for screening a compound useful for the prevention and / or treatment of normal-tension glaucoma using the knockout mouse.

Normal-tension glaucoma belongs to one type of glaucoma, but is a disease that has attracted particular attention recently because of its high prevalence. In general, curacia is a disease in which the optic nerve is compressed and atrophy because of high intraocular pressure (water pressure in the eyeball), resulting in impaired visual function and narrowed visual field. Finally, there is a high risk of blindness. On the other hand, normal-tension glaucoma is a pathological condition that exhibits the same findings (atrophy of the optic nerve and visual field loss) as glaucoma with high intraocular pressure, even though the intraocular pressure is in the normal range (usually 10 to 21 mmHg in humans). . Glaucoma is ranked second in diabetes in developed countries after diabetes, and about 2 million people, about 3.5% of people over 40 years old, are affected in Japan. Epidemiological studies have reported that 70% of them are normal-tension glaucoma. Normal-tension glaucoma progresses slowly and has few subjective symptoms, so early detection is difficult, and there is currently no conclusive treatment other than lowering intraocular pressure.
In recent years, degenerative loss of retinal ganglion cells, that is, neuronal death, induced by a mild and chronic increase in glutamate concentration has been proposed as one of the causes of glaucoma and diabetic retinopathy ((Harada, T Proc Natl Acad Sci USA 95, 4663-4666, 1998; Harada, C. et al., Neurosci. Lett. 292, 134-136, 2000).
In the mammalian central nervous system, glutamate is one of the major excitatory neurotransmitters and plays an important role in higher brain functions, whereas excessive glutamate is neurotoxic, It is known to cause various neurodegenerative diseases and delayed neuronal cell death after cerebral ischemia. One mechanism for regulating the glutamic acid concentration is a glutamate transporter. The glutamate transporter is a functional molecule whose main role is to take glutamate once released from nerve endings into cells and keep the glutamate concentration in the synaptic space low.
Currently, glutamate transporters in the mammalian brain include the types of EAAC1, EAAT4 and EAAT5 (Kanai, Y. & Heidiger, MA, Nature 360, 467-471, 1992) present in neurons. W.A., et al., Nature, 375, 599-603, 1995; Arizal, JE, et al., Proc Natl Acad Sci USA 94, 4155-4160, 1997, present in glial cells as well. Types of GLT1 and GLAST (also known as GluT-1) are known (Pines, D. et al., Nature 360, 464-467, 1992; Mukainaka et al., Biochimica et Biophysica Acta 12 Tanaka, K., Neurosci.Res. 16, 149-153, 1993; Tanaka, K., Neurosci. Lett.159, 183-186, 1993; Storck, T., et al. , Proc. Natl. Acad. Sci. USA 89, 10955-10959, 1992), and the relationship between abnormal function of these glutamate transporters and various neurodegenerative diseases is known.
Under such circumstances, GLAST was found to be a retina from an experiment using a GLAST knockout mouse (Watase, K. et al, Eur. J. Neurosci. 10, 976-988, 1998; JP-A-10-33087). In GLAST knockout mice, and the retinal damage after ischemic load is markedly exacerbated in GLAST knockout mice compared to the wild type. It has been suggested to be involved in the development of glaucoma (Harada, T., et al. Proc Natl Acad Sci USA 95, 4663-4666, 1998)). However, in this GLAST knockout mouse, damage to the retinal tissue has not been observed unless an ischemic load is applied, and this cannot be used as a normal-tension glaucoma model.
In order to develop therapeutic drugs for glaucoma and elucidate the mechanism of their development, genetic chronic glaucoma model mice and high-tension glaucoma model rabbits with water load exist as glaucoma model animals. No model animal has ever been known. In addition, there is no report pointing out the relationship between normal-tension glaucoma and GLAST, and the onset mechanism of normal-tension glaucoma remains unclear.
Therefore, if a normal-tension glaucoma model animal is obtained, the development of therapeutic agents effective for the treatment of the disease, the establishment of the treatment method, and the elucidation of the cause and pathogenesis of the disease Therefore, it is expected to be extremely useful. However, at present, a normal-tension glaucoma model animal is not known, and therefore, such a model animal has been eagerly desired in the medical field or the pharmaceutical field.

The present inventor has improved a conventional knockout mouse (GLAST knockout mouse) in which the function of the existing glutamate transporter gene GLAST is deficient. As a result, the retinal ganglion cell is in spite of the normal pressure within the normal range. Improved GLAST knockout mice with degenerate shedding and a marked decrease in the number of these can be obtained. This knockout mouse was found to be useful as a model mouse for normal-tension glaucoma.
Accordingly, the present invention relates to a GLAST knockout mouse as a model of normal-tension glaucoma that lacks the function of the endogenous GLAST gene, in particular, 1) its intraocular pressure is in the normal range, and 2) its retinal ganglion. Provides a GLAST knockout mouse in which the number of cells is reduced compared to wild type mice.
In the present invention, the intraocular pressure of the GLAST knockout mouse is usually 21 mmHg or less, for example, 10 to 21 mmHg. In addition, the number of cells in the retinal ganglion of the GLAST knockout mouse is reduced by at least 20% compared to the wild type mouse.
In the present invention, preferably, the genetic background of the GLAST knockout mouse is the same as or substantially the same as that of a C57BL / 6 mouse, for example, a C57BL / 6J mouse.
Specifically, the present invention provides a GLAST knockout mouse in which a neomycin resistance gene is inserted into the endogenous GLAST gene region, for example, in its sixth exon.
The present invention also provides the use of such a GLAST knockout mouse as a model mouse for normal tension glaucoma.
In another aspect, the present invention provides a method for producing a GLAST knockout mouse that lacks the function of the endogenous GLAST gene. This fabrication method comprises the following steps 1-6:
1) obtaining any mouse ES cell that lacks the function of one endogenous GLAST gene on a homologous chromosome;
2) Obtaining a chimeric mouse comprising the ES cell obtained in the step 1,
3) crossing the chimeric mouse obtained in step 2 with a normal C57BL / 6 mouse to obtain a heterozygous knockout mouse;
4) crossing the heterozygous knockout mouse obtained in step 3 with a normal C57BL / 6 strain mouse to obtain a heterozygous knockout mouse;
5) The mating described in Step 4 is repeated at least 5 times in total to obtain a heterozygous knockout mouse whose genetic background is close to that of C57BL / 6 strain mice, and 6) Heterozygous obtained in Step 5 Mating type knockout mice to obtain homozygous or heterozygous GLAST knockout mice.
In the production method of the present invention, in step 5, it is preferable to repeat the mating described in step 4 at least nine times in total.
A GLAST knockout mouse produced by the production method of the present invention is also included in the present invention, and the GLAST knockout mouse produced in this manner can also be used as a model mouse for normal tension glaucoma.
As yet another aspect, the present invention provides a method for using the above-mentioned GLAST knockout mouse of the present invention or the GLAST knockout mouse prepared by the above-described production method of the present invention as a model mouse for normal tension glaucoma. To do.
Therefore, the present invention provides a method for screening a compound useful for the prevention and / or treatment of normal-tension glaucoma using such a GLAST knockout mouse. In particular, this screening method is
1) administering a test compound to the GLAST knockout mouse of the present invention;
2) administering a test compound to a wild-type mouse,
3) In each of the above mice, evaluate the quantity or function of optic nerve cells surviving before administration and after a certain period of time after administration, and 4) compare the test results of knockout mice and wild type mice, Assessing the effectiveness of the test compound.
In the screening method of the present invention, in order to evaluate the number of living optic nerve cells or optic nerve cell function, in addition to counting the number of nerve cells in the retinal ganglion, measurement of retinal potential and visual evoked potential (Porcatitti et al., Vision Res. 39, 3071-3081, 1999), Visual Cliff test and the like (Ma, L. et al., Neuron 36, 623-634, 2002).

FIG. 1 shows an outline of the genomic structure of the mouse GLAST gene (restriction enzyme site and exon part). In the figure, the restriction enzymes involved in the restriction sites indicated by each symbol are as follows. E: EcoRI, B: BamHI. Moreover, a black box shows Exon (Exon) 1-10.
FIG. 2 shows the structure of the target gene region (upper) to be disrupted in order to delete the function of the mouse GLAST gene, the targeting vector (interruption) used, and the disrupted GLAST gene (lower) in Example 1. Show. In the figure, the restriction enzymes involved in the restriction sites indicated by each symbol are as follows. P: PvuII, RV: EcoRV, B: BamHI, E: EcoRI, X: XhoI. Further, exons 6 to 8 (E6 to 8) are indicated by black boxes. neo indicates a neomycin resistance gene, and DT-A indicates a diphtheria toxin A fragment gene.
FIG. 3 shows pathological slice images of the retina of homozygous GLAST knockout mice (GLAST − / −) and wild type normal mice (GLAST + / +).
FIG. 4 shows retinas of homozygous GLAST knockout mice (GLXST − / −), heterozygous GLAST knockout mice (GLAST +/−), and wild type normal mice (GLAST + / +) at the indicated age after birth. Shows the number of ganglion cells. These cell numbers were counted after hematoxylin / eosin staining of the number of neurons in the pathological section of the retinal ganglion. The vertical axis represents the average number of cells per slice obtained from 3 to 22 slices. The horizontal axis represents the age of each mouse after birth.
FIG. 5 shows fluorescence images showing neurons labeled by Fluoro-Gold retrograde labeling in retinal ganglia of homozygous GLAST knockout mice (GLAST − / −) and wild type normal mice (GLAST + / +). Show.

表１は、ＧＬＡＳＴ遺伝子のエキソンとイントロンの接合部位の配列を示す。エキソンの核酸配列は大文字で、イントロンの核酸配列は小文字で示している。
本発明において、グルタミン酸トランスポーターＧＬＡＳＴ遺伝子の機能の欠損とは、相同染色体上の１又は２つのＧＬＡＳＴ遺伝子座に存在する１又は２つの内在性ＧＬＡＳＴ遺伝子領域において、その構造をコードする領域中、例えばエキソン中に、変異を導入することにより、あるいはＧＬＡＳＴ遺伝子の発現に関与する領域中、例えば、プロモーター領域やイントロン領域中に、変異を導入することにより、機能的なＧＬＡＳＴが発現しないようにすること、あるいはＧＬＡＳＴ遺伝子の発現が恒常的に抑制されていることを意味している。いずれの場合にしろ、内在性の１又は２つのＧＬＡＳＴ遺伝子が生体内で実質的に機能していない状態を指す。従って、本発明において、ＧＬＡＳＴノックアウトマウスは、２つの内在性ＧＬＡＳＴ遺伝子の機能が欠損しているホモ接合型及び１つの内在性ＧＬＡＳＴ遺伝子の機能が欠損しているヘテロ接合型を包含するが、該遺伝子の機能欠損の効果の点からホモ接合型マウスが好ましい。
この様な遺伝子の機能の欠損を、公知のノックアウトマウスの作製方法、例えばジーンターゲッティング法により達成することができる。また、上記の変異の導入は、ＧＬＡＳＴ遺伝子領域内の塩基の置換、塩基の欠損、又はその領域内への塩基の挿入であってもよい。
本発明において、「遺伝的背景が同一又は実質的に同一」とは、比較するマウス間において、注目する遺伝子型（ＧＬＡＳＴ遺伝子型）以外の全ての遺伝子型が９９％以上同一であることを意味する。具体的には、実施例において、Ｆ１のＧＬＡＳＴヘテロ接合型ノックアウトマウスを９世代以上正常Ｃ５７ＢＬ／６系マウスと戻し交配し、１２９系統由来の遺伝子が全遺伝子の１％以下になったことを意味する。
１．本発明のＧＬＡＳＴノックアウトマウス
本発明は、相同染色体上の１又は２つの内在性ＧＬＡＳＴ遺伝子の機能が欠損した、正常眼圧緑内障モデルマウスとしてのＧＬＡＳＴノックアウトマウスを提供し、具体的には、１）その眼圧が正常範囲にあり、かつ２）その網膜神経節の細胞数が、野生型正常マウスに比べて減少している、ＧＬＡＳＴノックアウトマウスを提供する。特には、その遺伝的背景がＣ５７ＢＬ／６系マウス、例えばＣ５７ＢＬ／６Ｊ系マウスと同一又は実質的に同一であるものが好ましい。
野生型正常マウスの眼圧は、通常、１０〜２１ｍｍＨｇであるが、本発明のノックアウトマウスでもまた、その眼圧は正常範囲内にある。ただし、個体により、上記範囲を逸脱することもあるが、高眼圧といわれる範囲、例えば３０ｍｍＨｇ以上にまで達するものではない。マウスの眼圧は、例えば電子眼圧計を用いて、測定することができる。
また、本発明のノックアウトマウスでは、その網膜神経節の神経細胞数が、正常マウスに比べて、少なくとも２０％、より好ましくは少なくとも５０％ほど減少している。網膜神経節の神経細胞数の減少は、慣用的な組織化学的な手法により、例えば切片を用いたヘマトキシリン／エオシン染色により、顕微鏡下に測定することができる。
更に、本発明のノックアウトマウスでは、野生型正常マウスに比べて、光刺激による網膜の電位変化の一種であるｂ波の低下も認められている。ｂ波はＧＬＡＳＴが存在するミューラー細胞を含む網膜内層の活動電位を反映するものであり、ＧＬＡＳＴによるグルタミン酸濃度調節機構が、視覚伝達においても重要な役割を持つことを示唆する（Ｈａｒａｄａ，Ｔ．，ｅｔ ａｌ．Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ ９５，４６６３−４６６６，１９９８）。
この網膜神経節の神経細胞数の減少は、神経変性又は神経細胞死ととらえることができる。一般に、正常眼圧緑内障を含む緑内障では、視神経乳頭部の萎縮や網膜神経繊維の欠損が認められており、現在では眼圧依存性にせよ非依存性にせよ緑内障の最終病像は、網膜神経節細胞死であると考えられている。
従って、本発明のＧＬＡＳＴノックアウトマウスは、その眼の性状、すなわち眼圧が正常範囲にあること、及び網膜神経節の細胞数が減少していることを考慮すると、正常眼圧緑内障のモデルマウスとして使用することができる。
２．本発明のＧＬＡＳＴノックアウトマウスの作製
第２の態様として、本発明は、本発明のＧＬＡＳＴノックアウトマウスの作製方法を提供する。この方法は、下記過程１〜６を含んで成る：
１）相同染色体上の１つの内在性ＧＬＡＳＴ遺伝子の機能を欠損させた任意のマウスＥＳ細胞を得ること、
２）過程１で得られたＥＳ細胞を用いて、該細胞を含んで成るキメラマウスを得ること、
３）過程２で得られたキメラマウスを、野生型Ｃ５７ＢＬ／６系マウスと交配して、ヘテロ接合型ノックアウトマウスを得ること、
４）過程３で得られたヘテロ接合型ノックアウトマウスを野生型Ｃ５７ＢＬ／６系マウスと交配して、次世代のヘテロ接合型ノックアウトマウスを得ること、
５）過程４に記載した交配を、少なくとも合計５回繰り返して、その遺伝的背景をＣ５７ＢＬ／６系マウスに近づけたヘテロ接合型ノックアウトマウスを得ること、及び
６）過程５で得られたヘテロ接合型ノックアウトマウス同志を交配して、ホモ接合型又はヘテロ接合型ＧＬＡＳＴノックアウトマウスを得ること。
上記過程５において、交配を少なくとも合計９回繰り返すことが好ましい。
正常眼圧緑内障モデルとして使用することができる本発明のＧＬＡＳＴノックアウトマウスを作製するために、基本的には、公知のノックアウトマウス作製方法、例えばジーンターゲティング法やジーントラップ法を用いることができる。基本的なノックアウトマウスの作製方法は、ＧＬＡＳＴ遺伝子の破壊を達成することができ、生存・繁殖の能力を消失していないマウスが得られるものである限り、特に制限はない。ノックアウトマウスの作製方法に関しては、例えば、村松正實、山本雅編集、『実験医学別冊 新訂 遺伝子工学ハンドブック 改訂第３版』（１９９９年、羊土社発行）や八木健編集、『実験医学別冊ザ・プロトコールシリーズ ジーンターゲティングの最新技術』（２０００年、羊土社発行）を参照することができ、適宜、この発明の実施に応用することができる。
従って、まず、慣用的な方法により、例えばジーンターゲティング法に従って、上記過程１〜４を行うことができる。これらの過程は、本発明者らの研究グループによる特開平１０−３３０８７及びＷａｔａｓｅ，Ｋ．ｅｔ ａｌ，Ｅｕｒ．Ｊ．Ｎｅｕｒｏｓｃｉ．１０，９７６−９８８，１９９８にも開示されている。
従来、上記過程４で得られたヘテロ接合型ＧＬＡＳＴノックアウトマウスの雌雄を交配して得られたホモ接合型又はヘテロ接合型ＧＬＡＳＴノックアウトマウスが既に開示されているが、このタイプのＧＬＡＳＴノックアウトマウスでは、野生型正常マウスと比べて、網膜神経節の細胞数に実質的な変化は認められなかった。ただし、該ＧＬＡＳＴノックアウトの眼に対して生理食塩水を１５０ｃｍ Ｈ_２Ｏの圧力で６０分間注入することより、網膜を一過的な虚血状態にした後にのみ、網膜神経節の細胞数の減少が観察されていた（Ｈａｒａｄａ，Ｔ．，ｅｔ ａｌ．Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ ９５，４６６３−４６６６，１９９８）。
この様なことから、この様な従来のＧＬＡＳＴノックアウトマウスを、正常眼圧緑内障のモデルとして用いることはできない。
本発明の作製方法に従って、この様な従来のＧＬＡＳＴノックアウトマウスを更に改良することにより、先に示した正常眼圧緑内障たる本発明のＧＬＡＳＴノックアウトマウスを作製することができる。
以下に、標準的なノックアウトマウス作製方法であるジーンターゲティング法を例として、本発明のノックアウトマウスの作製方法を説明する。
過程１
（１）ターゲティングベクターの作製。
ジーンターゲティング法では、マウスＥＳ細胞内の染色体上のＧＬＡＳＴ遺伝子座を破壊するために、ターゲティングベクターを用いて、該遺伝子座に変異を導入する。
ＧＬＡＳＴ遺伝子の機能を欠損させるためには、ＧＬＡＳＴ遺伝子のいずれかの部分に、例えば１又は複数のエキソン部分に、塩基の欠失を生じさせ、点変異を導入し、又は他の遺伝子を挿入することができる。通常、内在性ＧＬＡＳＴ遺伝子が破壊されたＥＳ細胞をより容易に選択するために、選択マーカー遺伝子を挿入することが好ましい。
そのような遺伝子として、ポジティブ選別に用いるマーカー遺伝子、例えばネオマイシン（ｎｅｏ）耐性遺伝子を用いうる。このネオマイシン耐性遺伝子は、ネオマイシン類似体であるＧ４１８を用いることにより目的遺伝子の選別を可能にする。また、目的遺伝子を選別除去するためにネガティブ選別に用いるマーカー遺伝子を用いることもできる。このような遺伝子としては、例えば、チミジンキナーゼ（ｔｋ）遺伝子（選別剤としてガンシクロビル、ＦＩＡＵ等を用い、それに対する感受性により非相同組換え体を選別除去する）、ジフテリアトキシンＡフラグメント（ＤＴ−Ａ）遺伝子（ＤＴ−Ａにより発現されたジフテリア毒素により、非相同組換え体を選別除去する）が用いられる。あるいは、ポジティブ／ネガティブ選別を行うために、これらの組み合わせを用いることもできる。例えば、ネオマイシン耐性遺伝子及びジフテリアトキシンＡフラグメント遺伝子（Ｙａｇｉ，Ｎａｄａ，Ｗａｔａｎａｂｅｅｔ ａｌ．，Ａｎａｌｙｔｉｃａｌ Ｂｉｏｃｈｅｍｉｓｔｒｙ ２１４，７７−８６，１９９３）、あるいはネオマイシン耐性遺伝子及びチミジンキナーゼ遺伝子（Ｍａｎｓｏｕｒ，Ｔｈｏｍａｓ，Ｃａｐａｃｃｈｉ，Ｎａｔｕｒｅ ３３６，３４８−３５２，１９８８）を挿入することが好ましい。
該遺伝子配列中、変異を導入する部分、例えば上記マーカー遺伝子を挿入する部分は、該遺伝子の機能が欠損する部位であれば特に限定されないが、通常エキソン部分である。
ＧＬＡＳＴ遺伝子のゲノム構造（制限酵素地図及び各エキソン−イントロン連結点）が既に知られており（Ｈａｇｉｗａｒａ，Ｔ．，ｅｔ ａｌ．，Ｇｅｎｏｍｉｃｓ ３３，５０８−５１５，１９９６）、その構造の概略を図１及び表１に示す。マウスＧＬＡＳＴ遺伝子は１０個のエキソンを含み、いずれかのエキソンに、該遺伝子の欠損が生じるようにマーカー遺伝子を挿入することが好ましい。
上記の通りに該遺伝子の機能を破壊するために、標的遺伝子との相同組換が可能であり、その結果、標的遺伝子に変異を導入することができるターゲティングベクター（相同組換え用ＤＮＡ）を、ＧＬＡＳＴをコードする核酸配列（配列番号１）及びＧＬＡＳＴ遺伝子のゲノム配列の情報（Ｈａｇｉｗａｒａ，Ｔ．，ｅｔ ａｌ．，Ｇｅｎｏｍｉｃｓ ３３，５０８−５１５，１９９６）を基にして、常用のＤＮＡ組換え技術、例えばＰＣＲ法や部位特異的変異導入法により、作製することができる。
例えば、該遺伝子の全部又はその断片を含むＤＮＡ分子を、使用するＥＳ細胞の基となった系統のマウスから慣用的な方法によって単離する。該ＤＮＡ分子は、ＧＬＡＳＴ遺伝子の全長、若しくは全長とともに該遺伝子の５′上流域及び／又は３′下流域をさらに含むＤＮＡ分子であってもよい。
次に、得られたＤＮＡ分子から、該遺伝子中、変異を導入する部位に相当する部分に、所望の変異を導入した、例えば、上記のマーカー遺伝子を導入した改変ＤＮＡ分子を調製する。塩基配列の改変は、ＰＣＲにより増幅したＤＮＡ分子の連結や、部位特異的変異など慣用の組換えＤＮＡ技術によればよい。このようなターゲティングベクターを構築する際に、ターゲティングベクター構築用として市販されているプラスミドベクターを利用してもよい。
（２）ターゲティングベクターのＥＳ細胞への導入、及び内在性ＧＬＡＳＴ遺伝子との相同組換。
こうして得られたターゲティングベクターを、マウス胚性幹細胞（ＥＳ細胞）に導入し、ＥＳ細胞中のＧＬＡＳＴ遺伝子との間で相同組換えを行わせる。ターゲティングベクターのＥＳ細胞への導入は、例えばエレクトロポレーション法やリポフェクション法等の、慣用のＤＮＡ導入法により行うことができる。ターゲティングベクターが導入された細胞内では、その染色体上のＧＬＡＳＴ遺伝子とターゲティングベクター上の対応部分との間で相同組換えが生じ、その内在性遺伝子内に、ターゲティングベクター中の改変された塩基配列、例えばマーカー遺伝子が挿入される。その結果、ＥＳ細胞は内在性ＧＬＡＳＴ遺伝子の機能を欠損し、例えば同時にマーカー遺伝子を含むことになる。ターゲティングベクター導入後の細胞を、例えばマーカー遺伝子の選別機能により、あるいは相同的組換えを確認するサザンブロッティング法、ＰＣＲ法等の常法によりスクリーニングすることにより、ＧＬＡＳＴ遺伝子機能を欠損したＥＳ細胞（以下、組換えＥＳ細胞と称す）を得ることができる。なお一般的には、この様な相同組換により相同染色体の一方のみのＧＬＡＳＴ遺伝子が破壊されたＥＳ細胞が得られる。
使用するマウスＥＳ細胞としては、一般的には既に樹立されている１２９系のＥＳ細胞が用いられる。その他、Ｃ５７ＢＬ／６系やＢＤＦ１系（Ｃ５７ＢＬ／６系とＤＢＡ／２系との交配によるＦ１）マウスを用いて、公知の方法（Ｔｅｒａｔｏｃａｒｃｉｎｏｍａｓ ａｎｄ Ｅｍｂｒｙｏｎｉｃ Ｓｔｅｍ Ｃｅｌｌｓ：ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ（Ｒｏｂｅｒｔｓｏｎ，Ｅ．Ｊ．ｅｄ．），ＩＲＬ ｐｒｅｓｓ，Ｏｘｆｏｒｄ，１９８７）に従って樹立したＥＳ細胞を用いることもできる。好ましくは、１２９系のＥＳ細胞が用いられる。
過程２，３
Ｆ１世代のヘテロ接合型ＧＬＡＳＴ遺伝子ノックアウトマウスの作製
次に、得られた組換えＥＳ細胞を発生させてキメラマウスを得る。そのために、組換えＥＳ細胞を、マイクロインジェクション法や凝集法により、胚盤胞期又は８細胞期等の正常なマウス胚に注入し、その様にして得られたキメラ胚を、擬妊娠状態にある雌性マウスの子宮角に移植し、この移植マウスを通常通り飼育して、キメラマウス仔を出産させることができる。好ましくは、組換えＥＳ細胞を、Ｃ５７ＢＬ／６系マウスの胚に注入することが好ましい。
このキメラマウスは、通常、その体細胞及び生殖細胞として、組換えＥＳ細胞由来の細胞と正常細胞とを含んでなり、これを適当な系統の野生型マウス、好ましくはＣ５７ＢＬ／６系マウス、例えばＣ５７ＢＬ／６Ｊ系マウスと交配することによりヘテロ接合型のＦ１産仔が得られる。通常、雄性キメラマウスと雌性野生型マウスとを交配して、Ｆ１世代のヘテロ接合型マウスを産出させる。交配に用いたキメラマウスの生殖細胞が、上記の組換えＥＳ細胞、すなわち相同染色体の一方に存在する内在性ＧＬＡＳＴ遺伝子が破壊されているＥＳ細胞に由来していれば、該遺伝子の機能が欠損した所望のヘテロ接合型Ｆ１マウスを得ることができる。
上記行程において、ヘテロ接合型Ｆ１マウスを高効率に得るために、例えば、キメラ胚を作製する際に、組換えＥＳ細胞の起源マウスとは異なる体毛色のマウスに由来する正常宿主胚細胞を組み合わせれば、体毛色を観察することにより、生体における組換えＥＳ細胞の占める割合の高いキメラマウスやヘテロ接合型Ｆ１マウスの選択が容易となる。
Ｆ１世代において所期の遺伝子型が達成されているか否かは、その尾から抽出したＤＮＡに対して、サザンブロット法やＰＣＲ法により分析を行うことにより、確認することができる。
過程４，５，６
（１）本発明のＧＬＡＳＴ遺伝子ノックアウトマウスの獲得
本発明では、ＧＬＡＳＴ遺伝子ノックアウトマウスの遺伝的背景を、できるだけＣ５７ＢＬ／６系マウスに近づけることが好ましい。そのために、上記の通りに作製したＦ１ヘテロ接合型マウスを更にＣ５７ＢＬ／６系マウス、例えばＣ５７ＢＬ／６Ｊ系マウスと交配し、生まれてきたヘテロ接合型マウスを、再度Ｃ５７ＢＬ／６系野生型マウスと交配するという作業を繰り返す。この作業を、通常合計少なくとも５回、好ましくは少なくとも９回、より好ましくは少なくとも１５回行う。最後に、得られたヘテロ接合型マウスの雌雄を交配して、ＧＬＡＳＴ遺伝子の機能を欠損した、本発明のホモ接合型又はヘテロ接合型ノックアウトマウスを得ることができる。グルタミン酸トランスポーター遺伝子の機能欠損の効果の点からホモ接合型マウスが好ましい。
各世代において所期の遺伝子型が達成されているか否かは、上記と同様にサザンブロッティング法、ＰＣＲ法、塩基配列決定等の常法によればよい。
この様に作製することができる本発明のノックアウトマウスを、雄性・雌性の組合わせとして一旦得ておけば、それ以降は、必要に応じて適宜繁殖させることにより、容易に同じ遺伝子型のノックアウトマウスを必要数得ることができる。
（２）本発明のＧＬＡＳＴノックアウトマウスの網膜及び眼圧の解析
最後に、上記の通りに作製したホモ接合型又はヘテロ接合型ＧＬＡＳＴノックアウトマウスにおいて、その眼圧が正常範囲にあること、及び網膜神経節の神経細胞が減少していることを確認する。この様な眼の性状が確認できない場合には、過程５に記載の交配を更に繰り返すことによって、上記性状を満たす本発明のノックアウトマウスを得ることができる。
本発明のＧＬＡＳＴノックアウトマウスの眼圧は、通常約２１ｍｍＨｇ以下、例えば約１０〜２１ｍｍＨｇである。用いたＥＳ細胞の起源であるマウスの系統、又はキメラ胚の作製に用いた正常胚の起源であるマウスの系統によって、この眼圧範囲は多少変動しうるが、それを考慮しもて３０ｍｍＨｇ以下でなければならない。
本発明のＧＬＡＳＴノックアウトマウスでは、その網膜神経節の神経細胞数が、野生型マウスに比べて、少なくとも２０％、より好ましくは少なくとも５０％ほど減少している。
以下に、網膜神経節細胞数の計測方法と眼圧の測定方法の例を説明するが、これらの方法に限らず、公知の慣用的な方法を用いてよい。例えば、Ｈａｒａｄａ，Ｔ．，ｅｔ ａｌ．Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ ９５：４６６３−４６６６，１９９８やＨａｒａｄａ，Ｃ．ｅｔ ａｌ．，Ｎｅｕｒｏｓｃｉ．Ｌｅｔｔ．２９２，１３４−１３６，２０００を参照されたい。
これらの測定では、上記の本発明のホモ接合型又はヘテロ接合型ノックアウトマウスに対する対照マウスとして、それらのマウスを得る際に同時に出生した野生型正常マウス、あるいは単に正常（野生型）Ｃ５７ＢＬ／６系マウスを用いうる。
必要に応じて、上記の対照マウスの他に、上記のＦ１ヘテロ接合型ノックアウトマウス若しくはその雌雄を交配することにより得られるホモ接合型又はヘテロ接合型ＧＬＡＳＴノックアウトマウス、又は戻し交配の途中で得られるヘテロ接合型ノックアウトマウスなどにおいても、上記の測定を行う。
網膜神経節細胞数の計測：
マウスの網膜神経節細胞数を、通常の組織化学的な手法により、又は逆行性ラベリング法により測定することができる。
（ａ）病理切片を用いた方法
１）試験対象マウスを、麻酔により鎮静させ、４％パラホルムアルデヒド／ＰＢＳ溶液にて灌流固定する。
２）眼球を取り出して４℃の同液中で更に２時間固定する。
３）眼球をパラフィンに包埋した後、切片、例えば厚さ７μｍの切片を作製する。
４）ヘマトキシリン／エオシン染色を行い、顕微鏡下に、視神経を含む断面で神経節細胞数をカウントする。
（ｂ）逆行性ラベリングを用いた方法
１）麻酔により鎮静したマウスの頭部を固定する。
２）顕微鏡下で、エタノール噴霧した後、頭部をハサミで横切開し、頭蓋骨を露出させる。
３）縫合線と血管を確認後、グラインダーで手術用の穴をあけ、そこからマイクロシリンジで上丘実質に、例えば蛍光色素Ｆｌｕｏｒｏ−Ｇｏｌｄ（一般名ａｍｉｎｏｓｔｉｌｂａｍｉｄｉｎｅ；Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社）やカルボシアニン蛍光色素、例えばＤｉＩ（一般名１，１０−ｄｉｏｃｔａｄｅｃｙｌ−３，３，３０，３０−ｔｅｔｒａｍｅｔｈｙｌｉｎｄｏｃａｒｂｏｃｙａｎｉｎｅｐｅｒｃｈｌｏｒａｔｅ；Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅｓ社）を注入する。
４）皮膚をクリップで綴じた後、覚醒薬を腹腔内に注射して、回復を確認する。
５）手術後７日間通常通り飼育した後、処置マウスをエーテル麻酔にて死亡させ、眼球を取り出し、前眼部を除去する。
６）網膜を含む後眼部を４％パラホルムアルデヒド溶液に入れ、４℃で２０分間固定する。
７）網膜を取り出して伸展標本を作成する。
８）蛍光顕微鏡にて写真撮影後、蛍光ラベルされた網膜神経節細胞数をカウントする。
マウスの麻酔は、通常の動物実験に使用される麻酔剤であれば、マウスを死亡させない濃度範囲で、いずれのものを用いてもよい。例えばケタミン（１０ｍｇ／ｍｌ）／メデトミジン（１ｍｇ／ｍｌ）の１：１混合液（０．１５−０．２ｍｌ／マウス）を用いてよく、この場合アチパメゾール（５ｍｇ／ｍｌ）（０．１５−０．２ｍｌ／マウス）により覚醒させることができる。
眼圧の測定：
１）上記と同様に、通常の麻酔によりマウスを鎮静させる。
２）鎮静したマウスの眼圧を、電子眼圧計（例えばトノペンＸＬ、米国メドトロニック・ソーラン社製）を用いて測定する。
視覚機能又は視神経細胞の機能を評価するためには、網膜電図（Ｅｌｅｃｔｒｉｃｒｅｔｉｎｏｇｒａｍｓ，ＥＲＧ）を測定してもよい。これは、光刺激により網膜から得られる電気的反応を測定するものであり、生体における視神経細胞の神経伝達の活動度を表す、よい指標である。詳しくは、「現代の眼科学 改訂第８版」（所敬、金井淳編集、金原出版発行）や「視能矯正学 改訂第２版」（丸尾敏夫編集、金原出版発行）、又は非特許文献１（Ｈａｒａｄａ，Ｔ．，ｅｔ ａｌ．Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ ＵＳＡ ９５：４６６３−４６６６，１９９８）を参照されたい。
以下に、その測定方法を簡単に説明する。
１）マウスを常法により麻酔した後、頭部ホルダーを用いてマウスの位置を固定する。
２）０．５％フェニレフリン及び０．５％トロピカミドを投与して、瞳孔を拡張させる。
３）炭素繊維電極を角膜表面に接触させ、基準電極を前頭皮下に接触させる。
４）３０分間暗順応させる。
５）光刺激装置（ＳＬＳ−３１００，Ｎｉｈｏｎ Ｋｏｄｅｎ，Ｊａｐａｎ）により、０．６又は１．２Ｊの強度で、１０μ秒間の閃光を与えて、網膜を刺激する。
６）得られた電位を、増幅器（ＭＥＢ−５３０４，Ｎｉｈｏｎ Ｋｏｄｅｎ）により、周波数の帯域を５０−１０００Ｈｚ及び１−１０００Ｈｚ（各々振動電位ＯＰ及びａ波又はｂ波を測定する場合）に設定して、増幅する。
７）２回の応答電位を、平均化して記録する。
８）データとして、ａ波、ｂ波、及び各種振動電位を解析する。
３．正常眼圧緑内障の予防及び／又は治療に有用な化合物のスクリーニング方法
本発明のノックアウトマウスは、正常眼圧緑内障を予防及び／又は治療するために有効な化合物、特には網膜神経節の神経細胞を含む、視神経細胞の死もしくは変性、又はその機能の低下を抑制するために有効な化合物、あるいは視神経細胞又はその機能を回復させるために有効な化合物をスクリーニングするために用いることができる。
従って、本発明は、正常眼圧緑内障の予防及び／又は治療に有用な化合物のスクリーニング方法であって、
１）本発明のホモ接合型又はヘテロ接合型ＧＬＡＳＴノックアウトマウスに試験化合物を投与すること、
２）野生型正常マウスに試験化合物を投与すること、
３）上記の各マウスにおいて、投与前、及び投与してから一定期間後、生存する視神経細胞の数量又機能を評価すること、及び
４）ＧＬＡＳＴノックアウトマウスと野生型マウスの検査結果を比較して、試験化合物の有効性を評価すること
を含んで成るスクリーニング方法を提供する。
試験化合物が、正常眼圧緑内障の予防及び／又は治療にとって有効であるか否かは、緑内障に特徴的な徴候を改善できるか否かを検査することのよって判定することができる。例えば、網膜神経節の神経細胞数を計数することにより、試験化合物の投与により、対照マウスに比べて、その数が少なくとも１０％、好ましくは少なくとも２０％、より好ましくは少なくとも３０％回復していれば、その試験化合物は、医薬上有効であると判定してよい。また、網膜電位を測定することにより、試験化合物の投与により、対照値に比べて、例えばｂ波や振動電位の大きさが、少なくとも１０％、好ましくは少なくとも２０％、より好ましくは少なくとも３０％回復していれば、その試験化合物は、医薬上有効であると判定してよい。
本発明のヘテロ接合型ノックアウトマウスは、出生後、週齢と共に網膜神経節の細胞数が徐々に低下していくことが判明している（図４）。従って、上記スクリーニング方法の１つの態様として、１）出生後直ぐに、１群のヘテロ接合型ノックアウトマウスには定期的に試験化合物を投与し、もう１群のヘテロ接合型ノックアウトマウスには試験化合物を投与せず、２）各週齢で、網膜神経節の細胞数を測定し、そして３）両者の比較から、網膜神経節の細胞数の経時的な低下を抑制する化合物を選別することもできる。
試験化合物としては、天然及び合成化合物の他、動植物の抽出物、発酵生成物、ペプチド、蛋白質、又は核酸分子など、任意の物質を用いることができる。所望の蛋白質を発現するための遺伝子ベクターであってもよい。試験化合物の投与経路も、試験化合物の性質が許す限り、種々の方法を試みてよく、例えば点眼や経口投与によることもできる。投与期間や投与様式も、試験化合物の効果を最大限にする様に選択される。この様な試験化合物の種類や投与方法は、製薬分野又は医学分野における通常の方法に従ってもよい。
更に、本発明のＧＬＡＳＴノックアウトマウスを、他の種類のノックアウトマウス又は他の種類の疾患モデルマウスと交配して、新たな疾患モデルマウスを作製することもできる。この様な、本発明のＧＬＡＳＴノックアウトマウスの使用もまた本発明に含まれる。  In the present invention, the mouse glutamate transporter GLAST (Glutamate / Aspartate Transporter) is a protein encoded by a DNA chain having the base sequence shown in SEQ ID NO: 1 and having the amino acid sequence shown in SEQ ID NO: 2 (Tanaka) , K., Neurosci. Lett. 159, 1803-186, 1993)). This protein is also called GluT-1 in rats (Tanaka, K., Neurosci. Res. 16, 149-153, 1993; Stock, T., et al., Proc. Natl. Acad. Sci. USA). 89, 10955-10959, 1992), both are so-called counterparts.
  The gene (genome) structure of mouse GLAST has already been elucidated, details of which are described in Hagiwara, T .; , Et al. Genomics 33, 508-515, 1996. The outline of this gene structure is shown in Table 1 and FIG.
  However, depending on the strain of the mouse, the mouse GLAST may have the above-described coding nucleic acid sequence and amino acid sequence, and further its genomic sequence mutated within a range that maintains its function, for example, bases and amino acid residues. In the present invention, such a mutant, for example, in the above-described encoding nucleic acid sequence, for example, 1 to 10, preferably 1 to 5 bases may be substituted or deleted, added or inserted. Substitution, deletion, addition, or insertion variant, or a variant in which, for example, 1 to 10, preferably 1 to 5, amino acids are substituted, deleted, addition, or insertion in the above amino acid sequence Etc. are also included in the mouse GLAST.

  Table 1 shows the sequence of exon and intron junction sites of the GLAST gene. The exon nucleic acid sequence is shown in upper case, and the intron nucleic acid sequence is shown in lower case.
  In the present invention, the deficiency of the function of the glutamate transporter GLAST gene refers to one or two endogenous GLAST gene regions present in one or two GLAST gene loci on homologous chromosomes in a region encoding the structure, for example, Preventing functional GLAST from being expressed by introducing mutations into exons or by introducing mutations into regions involved in GLAST gene expression, for example, promoter regions or intron regions Or it means that the expression of the GLAST gene is constantly suppressed. In any case, it refers to a state in which one or two endogenous GLAST genes are not substantially functioning in vivo. Therefore, in the present invention, GLAST knockout mice include a homozygous type in which the functions of two endogenous GLAST genes are defective and a heterozygous type in which the function of one endogenous GLAST gene is defective. Homozygous mice are preferable from the viewpoint of the effect of gene function loss.
  Such a deficiency in gene function can be achieved by a known method for producing a knockout mouse, for example, a gene targeting method. Moreover, the introduction of the mutation may be a base substitution in the GLAST gene region, a base deletion, or a base insertion into the region.
  In the present invention, “the genetic background is the same or substantially the same” means that all genotypes other than the genotype of interest (GLAST genotype) are 99% or more identical among the mice to be compared. To do. Specifically, in the Examples, F1 GLAST heterozygous knockout mice were backcrossed with 9 generations or more of normal C57BL / 6 mice, meaning that the genes derived from the 129 strain were 1% or less of the total genes. To do.
1. GLAST knockout mouse of the present invention
  The present invention provides a GLAST knockout mouse as a normal-tension glaucoma model mouse that lacks the function of one or two endogenous GLAST genes on a homologous chromosome. Specifically, 1) the intraocular pressure is in the normal range. And 2) providing a GLAST knockout mouse in which the number of cells in the retinal ganglion is reduced compared to a wild type normal mouse. In particular, the genetic background is preferably the same or substantially the same as that of a C57BL / 6 mouse, for example, a C57BL / 6J mouse.
  The intraocular pressure of wild-type normal mice is usually 10 to 21 mmHg, but the intraocular pressure is also in the normal range in the knockout mouse of the present invention. However, although it may deviate from the above range depending on the individual, it does not reach a range called high intraocular pressure, for example, 30 mmHg or more. The intraocular pressure of the mouse can be measured, for example, using an electronic tonometer.
  Further, in the knockout mouse of the present invention, the number of nerve cells in the retinal ganglion is reduced by at least 20%, more preferably at least 50%, compared to the normal mouse. The decrease in the number of nerve cells in the retinal ganglion can be measured under a microscope by a conventional histochemical method, for example, by hematoxylin / eosin staining using a section.
  Furthermore, in the knockout mouse of the present invention, a decrease in b-wave, which is a kind of retinal potential change caused by light stimulation, is recognized as compared with a wild type normal mouse. The b wave reflects the action potential of the inner layer of the retina including the Müller cell in which GLAST is present, suggesting that the mechanism for regulating glutamate concentration by GLAST also plays an important role in visual transmission (Harada, T.,). et al. Proc Natl Acad Sci USA 95,4663-4666, 1998).
  This decrease in the number of nerve cells in the retinal ganglion can be regarded as neurodegeneration or nerve cell death. In general, glaucoma, including normal-tension glaucoma, has atrophy of the optic nerve head and deficits in retinal nerve fibers.Currently, the final image of glaucoma, whether dependent on intraocular pressure or independent, is It is thought to be node cell death.
  Therefore, the GLAST knockout mouse of the present invention is a normal intraocular pressure glaucoma model mouse in consideration of the properties of the eye, that is, the intraocular pressure is in the normal range and the number of cells of the retinal ganglion is reduced. Can be used.
2. Production of GLAST knockout mice of the present invention
  As a second aspect, the present invention provides a method for producing the GLAST knockout mouse of the present invention. This method comprises the following steps 1-6:
  1) obtaining an arbitrary mouse ES cell in which the function of one endogenous GLAST gene on a homologous chromosome is deleted;
  2) Obtaining a chimeric mouse comprising the ES cell obtained in the step 1,
  3) crossing the chimeric mouse obtained in step 2 with a wild type C57BL / 6 mouse to obtain a heterozygous knockout mouse;
  4) crossing the heterozygous knockout mouse obtained in step 3 with a wild type C57BL / 6 mouse to obtain a next generation heterozygous knockout mouse;
  5) Repeating the mating described in step 4 at least 5 times in total to obtain a heterozygous knockout mouse whose genetic background is close to that of C57BL / 6 strain mice, and
  6) Crossing heterozygous knockout mice obtained in step 5 to obtain homozygous or heterozygous GLAST knockout mice.
  In the above process 5, it is preferable to repeat the mating at least 9 times in total.
  In order to produce the GLAST knockout mouse of the present invention that can be used as a normal-tension glaucoma model, basically, a known knockout mouse production method such as a gene targeting method or a gene trap method can be used. The basic method for producing a knockout mouse is not particularly limited as long as the GLAST gene can be disrupted and a mouse that does not lose its ability to survive and reproduce can be obtained. Regarding the method of producing the knockout mouse, for example, Masaaki Muramatsu, Masaru Yamamoto, “Experimental Medicine Separate Volume, New Genetic Engineering Handbook, Third Edition” (1999, published by Yodosha), Ken Yagi, “Experimental Medicine, Separate Volume The Protocol Series The latest technology of gene targeting ”(2000, published by Yodosha) can be appropriately applied to the implementation of the present invention.
  Therefore, first, the above steps 1 to 4 can be performed by a conventional method, for example, according to the gene targeting method. These processes are described in JP-A-10-33087 and Watase, K. et al. et al, Eur. J. et al. Neurosci. 10, 976-988, 1998.
  Conventionally, a homozygous or heterozygous GLAST knockout mouse obtained by mating male and female of the heterozygous GLAST knockout mouse obtained in the above process 4 has already been disclosed, but in this type of GLAST knockout mouse, Compared to wild-type normal mice, there was no substantial change in the number of retinal ganglion cells. However, saline is applied to the eye of the GLAST knockout at 150 cm H₂A decrease in the number of cells in the retinal ganglion has been observed only after the retina has been brought into a transient ischemic state by injecting at a pressure of O for 60 minutes (Harada, T., et al. Proc Natl Acad Sci USA 95, 4663-4666, 1998).
  For this reason, such a conventional GLAST knockout mouse cannot be used as a model for normal-tension glaucoma.
  By further improving such a conventional GLAST knockout mouse according to the production method of the present invention, the GLAST knockout mouse of the present invention which exhibits normal-tension glaucoma described above can be produced.
  The method for producing the knockout mouse of the present invention will be described below by taking the gene targeting method, which is a standard method for producing knockout mice, as an example.
  Process 1
  (1) Preparation of targeting vector.
  In the gene targeting method, in order to destroy the GLAST locus on the chromosome in mouse ES cells, a mutation is introduced into the locus using a targeting vector.
  In order to delete the function of the GLAST gene, a base deletion is caused in any part of the GLAST gene, for example, in one or a plurality of exon parts, a point mutation is introduced, or another gene is inserted. be able to. Usually, in order to more easily select ES cells in which the endogenous GLAST gene is disrupted, it is preferable to insert a selection marker gene.
  As such a gene, a marker gene used for positive selection, for example, a neomycin (neo) resistance gene can be used. This neomycin resistance gene enables selection of a target gene by using G418, which is a neomycin analog. In addition, a marker gene used for negative selection to select and remove a target gene can also be used. Such genes include, for example, thymidine kinase (tk) gene (gancyclovir, FIAU, etc. are used as a selection agent, and non-homologous recombinants are removed by sensitivity to them), diphtheria toxin A fragment (DT-A) A gene (selective removal of non-homologous recombinants by diphtheria toxin expressed by DT-A) is used. Alternatively, a combination of these can be used for positive / negative selection. For example, the neomycin resistance gene and diphtheria toxin A fragment gene (Yagi, Nada, Watanabe et al., Analytical Biochemistry 214, 77-86, 1993), or the neomycin resistance gene and thymidine kinase gene (Mansour, Thomas, Capacchi, Nature 3 48). -352, 1988) is preferably inserted.
  In the gene sequence, the part into which the mutation is introduced, for example, the part into which the marker gene is inserted is not particularly limited as long as it is a part lacking the function of the gene, but is usually an exon part.
  The genomic structure of the GLAST gene (restriction enzyme map and each exon-intron junction) is already known (Hagiwara, T., et al., Genomics 33, 508-515, 1996). And in Table 1. The mouse GLAST gene contains 10 exons, and it is preferable to insert a marker gene in any exon so that the gene is defective.
  As described above, in order to destroy the function of the gene, homologous recombination with the target gene is possible, and as a result, a targeting vector (DNA for homologous recombination) capable of introducing a mutation into the target gene, Based on the nucleic acid sequence encoding GLAST (SEQ ID NO: 1) and information on the genomic sequence of the GLAST gene (Hagiwara, T., et al., Genomics 33, 508-515, 1996), For example, it can be prepared by PCR or site-directed mutagenesis.
  For example, a DNA molecule containing all of the gene or a fragment thereof is isolated by a conventional method from a mouse of the strain on which the ES cell used is based. The DNA molecule may be a full-length GLAST gene, or a DNA molecule that further includes the 5 ′ upstream region and / or the 3 ′ downstream region of the gene together with the full length.
  Next, from the obtained DNA molecule, a modified DNA molecule into which a desired mutation is introduced, for example, the above-described marker gene is introduced into a portion corresponding to the site into which the mutation is introduced in the gene is prepared. The base sequence can be modified by conventional recombinant DNA techniques such as ligation of DNA molecules amplified by PCR and site-specific mutation. When constructing such a targeting vector, a plasmid vector that is commercially available for constructing a targeting vector may be used.
(2) Introduction of targeting vector into ES cells and homologous recombination with endogenous GLAST gene.
  The targeting vector thus obtained is introduced into mouse embryonic stem cells (ES cells), and homologous recombination is performed with the GLAST gene in the ES cells. Introduction of the targeting vector into ES cells can be performed by a conventional DNA introduction method such as electroporation method or lipofection method. In the cell into which the targeting vector has been introduced, homologous recombination occurs between the GLAST gene on the chromosome and the corresponding part on the targeting vector, and within the endogenous gene, the modified base sequence in the targeting vector, For example, a marker gene is inserted. As a result, ES cells lack the function of the endogenous GLAST gene and, for example, simultaneously contain a marker gene. The cells after introduction of the targeting vector are screened by, for example, a marker gene selection function, or a conventional method such as Southern blotting method or PCR method for confirming homologous recombination. , Referred to as recombinant ES cells). In general, such homologous recombination provides an ES cell in which only one of the homologous chromosomes is disrupted.
  As the mouse ES cells to be used, 129 ES cells that have already been established are generally used. In addition, using a C57BL / 6 strain or a BDF1 strain (F1 by crossing between C57BL / 6 strain and DBA / 2 strain) mouse, a known method (Teratocarcinomas and Embryonic Stem Cells: a Practical Approach (Robert. ed.), IRL press, Oxford, 1987). Preferably, 129 type ES cells are used.
  Process 2 and 3
  Generation of F1 generation heterozygous GLAST gene knockout mice
  Next, the resulting recombinant ES cells are generated to obtain chimeric mice. Therefore, recombinant ES cells are injected into normal mouse embryos at the blastocyst stage or 8-cell stage by microinjection or aggregation, and the chimeric embryos thus obtained are put into a pseudopregnant state. A female mouse can be transplanted into the uterine horn, and the transplanted mouse can be bred as usual to give birth to a chimeric mouse offspring. Preferably, it is preferable to inject recombinant ES cells into embryos of C57BL / 6 mice.
  This chimeric mouse usually comprises a recombinant ES cell-derived cell and a normal cell as its somatic cell and germ cell, and this is a wild-type mouse of an appropriate strain, preferably a C57BL / 6 mouse, for example, Heterozygous F1 offspring can be obtained by crossing with C57BL / 6J mice. Usually, male chimeric mice and female wild-type mice are mated to produce F1 generation heterozygous mice. If the germ cell of the chimeric mouse used for mating is derived from the above recombinant ES cell, that is, an ES cell in which the endogenous GLAST gene present in one of the homologous chromosomes is disrupted, the function of the gene is defective. The desired heterozygous F1 mouse can be obtained.
  In the above process, in order to obtain a heterozygous F1 mouse with high efficiency, for example, when producing a chimeric embryo, a normal host embryo cell derived from a mouse with a hair color different from that of the mouse that originated the recombinant ES cell is combined. Thus, by observing the color of the body hair, it becomes easy to select chimeric mice and heterozygous F1 mice in which the proportion of recombinant ES cells in the living body is high.
  Whether or not the desired genotype has been achieved in the F1 generation can be confirmed by analyzing the DNA extracted from the tail by Southern blotting or PCR.
  Process 4, 5, 6
  (1) Acquisition of the GLAST gene knockout mouse of the present invention
  In the present invention, the genetic background of the GLAST gene knockout mouse is preferably as close as possible to the C57BL / 6 mouse. For this purpose, the F1 heterozygous mouse prepared as described above is further crossed with a C57BL / 6 mouse, for example, a C57BL / 6J mouse, and the heterozygous mouse thus born is again a C57BL / 6 wild-type mouse. Repeat the work of mating. This operation is usually performed a total of at least 5 times, preferably at least 9 times, more preferably at least 15 times. Finally, the resulting heterozygous mouse can be mated to obtain the homozygous or heterozygous knockout mouse of the present invention lacking the function of the GLAST gene. Homozygous mice are preferred from the viewpoint of the effect of functional loss of the glutamate transporter gene.
  Whether or not the desired genotype is achieved in each generation may be determined by conventional methods such as Southern blotting, PCR, and nucleotide sequencing as described above.
  Once the knockout mouse of the present invention that can be produced in this way is obtained as a combination of male and female, then the subsequent knockout mouse of the same genotype can be easily bred as needed. You can get as many as you need.
  (2) Analysis of retina and intraocular pressure of the GLAST knockout mouse of the present invention
  Finally, in the homozygous or heterozygous GLAST knockout mouse prepared as described above, it is confirmed that the intraocular pressure is in the normal range and that the nerve cells in the retinal ganglion are reduced. When such eye properties cannot be confirmed, the knockout mouse of the present invention satisfying the above properties can be obtained by further repeating the mating described in Step 5.
  The intraocular pressure of the GLAST knockout mouse of the present invention is usually about 21 mmHg or less, for example, about 10 to 21 mmHg. This intraocular pressure range may vary somewhat depending on the mouse strain that is the origin of the ES cells used, or the mouse strain that is the origin of the normal embryo used for the production of the chimeric embryo. Must.
  In the GLAST knockout mouse of the present invention, the number of neurons in the retinal ganglion is reduced by at least 20%, more preferably at least 50%, compared to the wild type mouse.
  Hereinafter, examples of a method for measuring the number of retinal ganglion cells and a method for measuring intraocular pressure will be described. However, the present invention is not limited to these methods, and a known conventional method may be used. For example, Harada, T .; , Et al. Proc Natl Acad Sci USA 95: 4663-4666, 1998 and Harada, C.I. et al. , Neurosci. Lett. 292, 134-136, 2000.
  In these measurements, as a control mouse for the above-mentioned homozygous or heterozygous knockout mouse of the present invention, a wild-type normal mouse born at the time of obtaining these mice, or simply a normal (wild-type) C57BL / 6 strain A mouse can be used.
  If necessary, in addition to the control mouse, a homozygous or heterozygous GLAST knockout mouse obtained by mating the F1 heterozygous knockout mouse or male and female, or obtained during backcrossing The above measurement is also performed in a heterozygous knockout mouse or the like.
  Retinal ganglion cell count:
  Mouse retinal ganglion cell numbers can be measured by conventional histochemical techniques or by retrograde labeling.
  (A) Method using pathological section
    1) Test mice are sedated by anesthesia and fixed by perfusion with 4% paraformaldehyde / PBS solution.
    2) The eyeball is taken out and fixed in the same solution at 4 ° C. for 2 hours.
    3) After embedding the eyeball in paraffin, a section, for example, a section having a thickness of 7 μm is prepared.
    4) Hematoxylin / eosin staining is performed, and the number of ganglion cells is counted in a cross section including the optic nerve under a microscope.
  (B) Method using retrograde labeling
    1) The head of a mouse sedated by anesthesia is fixed.
    2) After spraying with ethanol under a microscope, the head is transected with scissors to expose the skull.
    3) After confirming the suture line and the blood vessel, a surgical hole is made with a grinder, and from there, with a microsyringe into the upper hill parenchyma, for example, a fluorescent dye Fluoro-Gold (generic name: aminostylamidine; Molecular Probes), a carbocyanine fluorescent dye, For example, DiI (general name 1,10-dioctadecyl-3,3,30,30-tetramethyldocarbocyanine perchlorate; Molecular Probes) is injected.
    4) After binding the skin with a clip, a stimulant is injected into the abdominal cavity to confirm recovery.
    5) After normal operation for 7 days after the operation, the treated mouse is killed by ether anesthesia, the eyeball is taken out, and the anterior eye segment is removed.
    6) Place the posterior eye part including the retina in 4% paraformaldehyde solution and fix at 4 ° C. for 20 minutes.
    7) Remove the retina and create an extension specimen.
    8) Count the number of fluorescently labeled retinal ganglion cells after taking a picture with a fluorescence microscope.
  As long as the anesthesia of the mouse is an anesthetic used in normal animal experiments, any anesthetic may be used in a concentration range that does not cause the mouse to die. For example, a 1: 1 mixture of ketamine (10 mg / ml) / medetomidine (1 mg / ml) (0.15-0.2 ml / mouse) may be used, in which case atipamezole (5 mg / ml) (0.15-0 .2 ml / mouse).
  Measurement of intraocular pressure:
  1) As described above, the mouse is sedated by normal anesthesia.
  2) The intraocular pressure of the sedated mouse is measured using an electronic tonometer (for example, Tonopen XL, manufactured by Medtronic Solan, USA).
  In order to evaluate the visual function or the function of the optic nerve cell, electroretinogram (ERG) may be measured. This measures the electrical response obtained from the retina by light stimulation, and is a good index that represents the degree of nerve transmission activity of optic nerve cells in the living body. For details, see "Contemporary Ophthalmology Revision 8th Edition" (Tomaki, edited by Kei Kanai, published by Kanbara Publishing) or "Vision Corrections Revised 2nd Edition" (edited by Toshio Maruo, published by Kanehara Publishing) 1 (Harada, T., et al. Proc Natl Acad Sci USA 95: 4663-4666, 1998).
  The measurement method will be briefly described below.
  1) Anesthetize the mouse by a conventional method, and then fix the position of the mouse using a head holder.
  2) Administer 0.5% phenylephrine and 0.5% tropicamide to dilate the pupil.
  3) A carbon fiber electrode is brought into contact with the corneal surface, and a reference electrode is brought into frontal subcutaneous contact.
  4) Allow dark adaptation for 30 minutes.
  5) Using a light stimulator (SLS-3100, Nihon Koden, Japan), stimulate the retina by applying a flash of 10 μs at an intensity of 0.6 or 1.2 J.
  6) Set the obtained potential to 50-1000 Hz and 1-1000 Hz (when measuring vibration potential OP and a wave or b wave, respectively) with an amplifier (MEB-5304, Nihon Koden). Amplify.
  7) The two response potentials are averaged and recorded.
  8) As data, a wave, b wave, and various vibration potentials are analyzed.
3. Method for screening compound useful for prevention and / or treatment of normal-tension glaucoma
  The knockout mouse of the present invention suppresses death or degeneration of an optic nerve cell, or a decrease in its function, including a compound effective for preventing and / or treating normal tension glaucoma, particularly a nerve cell of a retinal ganglion. Therefore, it can be used for screening a compound effective for the purpose, or a compound effective for restoring the optic nerve cell or its function.
  Therefore, the present invention is a method for screening a compound useful for the prevention and / or treatment of normal tension glaucoma,
  1) administering a test compound to the homozygous or heterozygous GLAST knockout mouse of the present invention;
  2) administering a test compound to wild-type normal mice;
  3) In each of the above mice, evaluating the quantity or function of viable optic nerve cells before administration and after a certain period of time after administration; and
  4) To compare the test results of GLAST knockout mice and wild type mice to evaluate the effectiveness of the test compound.
A screening method comprising:
  Whether a test compound is effective for the prevention and / or treatment of normal-tension glaucoma can be determined by examining whether it can ameliorate symptoms characteristic of glaucoma. For example, by counting the number of neurons in the retinal ganglion, administration of the test compound can restore that number by at least 10%, preferably at least 20%, more preferably at least 30% compared to control mice. For example, the test compound may be determined to be pharmaceutically effective. In addition, by measuring the retinal potential, the magnitude of the b wave or the vibration potential is restored by at least 10%, preferably at least 20%, more preferably at least 30% compared to the control value by administration of the test compound. If so, the test compound may be determined to be pharmaceutically effective.
  In the heterozygous knockout mouse of the present invention, it has been found that the number of retinal ganglion cells gradually decreases with age after birth (FIG. 4). Therefore, as one aspect of the screening method, 1) a test compound is periodically administered to a group of heterozygous knockout mice immediately after birth, and a test compound is administered to another heterozygous knockout mouse. Without administration, 2) the number of cells in the retinal ganglion is measured at each week of age, and 3) a compound that suppresses the decrease in the number of cells in the retinal ganglion over time can be selected from the comparison between the two.
  As a test compound, in addition to natural and synthetic compounds, any substance such as animal and plant extracts, fermentation products, peptides, proteins, or nucleic acid molecules can be used. It may be a gene vector for expressing a desired protein. As for the route of administration of the test compound, various methods may be tried as long as the properties of the test compound allow, for example, eye drops or oral administration. The duration and mode of administration are also selected to maximize the effect of the test compound. Such test compound types and administration methods may be in accordance with conventional methods in the pharmaceutical or medical fields.
  Furthermore, the GLAST knockout mouse of the present invention can be crossed with another kind of knockout mouse or another kind of disease model mouse to produce a new disease model mouse. Such use of the GLAST knockout mouse of the present invention is also included in the present invention.

EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.
Example 1: Preparation of GLAST (GluT-1) gene function deficient mice (1) Preparation of DNA for homologous recombination of mouse GLAST (GluT-1) gene DNA Genomic DNA extracted from liver of 129SV mice (wild type mouse GLAST The genomic library lambda FIXII obtained by partial digestion of (GluT-1) gene with the restriction enzyme Sau3AI is hybridized using the partial sequence of the mouse GLAST (GluT-1) gene cDNA as a probe. As a result of searching for ⁶ colonies, 26 positive clones were obtained. This was incompletely digested with restriction enzymes EcoRV and XhoI, and subcloned into a full-length 9 kbp genomic DNA containing exons 6 to 8 (FIG. 2, upper panel). Next, in order to disrupt the GLAST (GluT-1) gene, the 1.5 kbp after BamHI of the 6th exon is deleted, the neomycin resistance gene is inserted therein, and the diphtheria toxin A fragment gene is further inserted downstream of the 8th exon. (FIG. 2, middle). The homologous part with the genomic DNA was constructed so that the upstream of the neomycin resistance gene was 2.5 kb, and the distance between the neomycin fertile gene and the diphtheria toxin A fragment gene was 5 kb. The resulting construct was inserted into pBluescriptSK, and when introduced into ES cells, it was cleaved with restriction enzyme NotI and linearized to obtain a targeting vector (DNA for homologous recombination: pGluT1NeoDT) (FIG. 2). , Interruption).
(2) Deletion of GLAST (GluT-1) gene of ES cell by introduction of DNA for homologous recombination 75 μg of DNA for homologous recombination contains buffer solution for electroporation (137 mM NaCl) containing 3 × 10 ⁷ mouse ES cells (E14 strain). , 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ ), and gene transfer was performed under the conditions of Field Strength 210 V / cm and Capacitance 500 μF. From 24 hours after the introduction, selective culturing was performed with 250 μg / ml G418 (Geneticin, GIBCOBRL).
A 96-well microplate containing 60 μl of Tris-EDTA solution (solution consisting of 10 mM Tris-HCl pH 8.0 and 1 mM EDTA pH 8.0) using a micropipette from 192 hours after gene introduction of G418 resistant colonies After transferring to (FALCON 3077) and treating for several minutes, the cells were pipetted into single cells, which were transferred to a 24-well microplate (FALCON 3047), and the culture was continued. The collected colonies had a major axis that reached 1/2 or more of the inner diameter of the microchip, and the number of cells at this time was 1 × 10 ⁴ to 10 ⁵ . The number of viable cells after electroporation was 6.0 × 10 ⁷ cells. The number of G418-resistant colonies was 2.4 × 10 ² , which was 1 / 2.5 × 10 ⁵ of the number of viable cells.
When cells on a 24-well microplate reached confluence in 3-4 days of culture, the cells were treated with 0.25% trypsin for 5 minutes at 37 ° C., and then 35 mm (FALCON 3001) or 60 mm ( The cells were cultured in a FALCON 3002) petri dish for tissue culture to proliferate the cells. All ES cell cultures were performed on feeder cells. Confirmation of homologous recombinants was performed by Southern blot as follows.
For Southern blot analysis, genomic DNA was extracted from G418-resistant cells, digested with the restriction enzyme PvuII, and then used with the ApaI-EcoRV fragment of the fifth intron, 0.5 kb as a probe. Confirmation of homologous recombinants (FIG. 2, bottom) and non-homologous recombinants containing the disrupted allele was performed by detection of 4.2 kb and 7 kb bands, respectively. The number of homologous recombinant colonies was 1 out of 242 G418 resistant colonies (2B7).
(3) ES cell and its culture method As ES cell, E14 strain derived from 129 / SvJ strain mouse blastocyst was used. For the culture of ES cells, Dulbecco's modified Eagle medium (DMEM, 11960-010 GIBCO), 15% fetal calf serum (FCS), 0.1 mM 2-mercaptoethanol, nucleic acid mixture, non-essential amino acid solution and 10 ³ An SCM culture solution (Robertson, Teratocarcinomas and embryonic cells and a clinical approach 1987) supplemented with unit / ml LIF (AMRAD) was used.
In addition, mouse embryo fibroblasts used as ES cell feeder cells were cultured in DMEM supplemented with 10% FSC. Mouse fetal fibroblasts were prepared and cultured as follows. After aseptically collecting fetuses of ICR mice of 13-14 days of gestation, washing with phosphate buffered saline (PBS-) without calcium and magnesium, the heart, liver and intestinal tract were removed using tweezers, Shred using ophthalmic scissors. Subsequently, the obtained fine slice was treated with PBS- (hereinafter referred to as TE solution) containing 0.25% trypsin and 0.04% EDTA at room temperature for 20 minutes to obtain a cell suspension.
After centrifuging the cell suspension at 1500 rpm for 5 minutes, the supernatant was removed, suspended in DMEM with 10% FCS, and allowed to stand for 2 minutes. Then, the cell suspension from which the tissue piece that had settled at the bottom was removed was transferred to a 100 × 20 mm tissue culture petri dish (FALCON 3003) and subjected to culture under conditions of 37 ° C., 5% CO ₂ , and 95% air. The next day, the cell suspension was washed once with PBS- and the culture was continued. Passaging was performed at intervals of 3 to 4 days, and mitomycin treatment was performed to use cells up to the third passage as feeder cells.
Mouse embryo fibroblasts grown to confluence were treated with 75 μl of 2 mg / ml mitomycin C for 3-4 hours, washed 3 times with PBS−, and then treated with TE solution at room temperature for 3 minutes to detach the cells. Subsequently, after centrifugation, the number of cells was adjusted to ⁵ × 10 ⁵ / ml, and 3 ml each was dispensed into a 60 × ¹⁰ mm gelatin-coated dish (FALCON 3002). The feeder cells prepared as described above were used within one week. ES cells were passaged by treating with TE solution for 5 minutes at room temperature, dispersing ES cells into single cells by pipetting, and seeding ⁴ × 10 ⁵ cells on the feeder cell layer.
The culture solution was changed every 24 hours, and the passage time was 56 to 64 hours. For cryopreservation, 1 × 10 ⁶ cells were suspended in SCM, transferred to a freezing tube (2 ml, FALCON 4818), and 0.5 ml of freezing medium (DMEM with 20% DMSO) was added dropwise. , Left at −80 ° C. overnight and stored in liquid nitrogen.
(4) Preparation of chimeric mice using GLAST (GluT-1) gene-deficient ES cells (a) Injection of GLAST (GluT-1) gene-deficient ES cells into blastocysts ES cells were blastocysts of C57BL / 6J mice After injection into the cyst, the resulting host embryo was transplanted into the uterine horn of a pseudopregnant mouse to obtain a litter. Host embryos were collected by perfusing the uterus with Hepes-buffered-Whitten's medium on the fourth day of natural mating. ES cells used for injection were treated with TE solution on the 2nd or 3rd day of passage, and then left to stand in a gelatin-coated dish for 30 minutes until the feeder cells were removed and subjected to microscopic manipulation. And left on ice.
The pipette for injecting ES cells is a fine glass tube (NARISHIGE) having an outer diameter of 1 mm, which is finely stretched using a microelectrode preparation device (NARISHIGE, PN-3), and the inner diameter is about 20 μm with a polishing machine (NARISHIGE). The tip was polished, and the tip was sharpened with a microforge (De Fombrun). The pipette for embryo retention was used after cutting the glass tube stretched by the above-mentioned method at a part having an outer diameter of 50 to 100 μm using a microforge and further processing the diameter to 10 to 20 μm.
The injection pipette and the holding pipette were connected to a micromanipulator (LEITZ) by bending a portion of about 5 mm from the tip thereof by about 30 degrees. The chamber used for the microscopic operation was a perforated slide glass with a cover glass adhered with beeswax, and about 20 μl of 5% FCS-added Hepes-buffered-Whitten's medium drop was placed on it. The upper surface was covered with mineral oil (M8410, Sigma). About 100 ES cells were placed in one drop, 10-15 expanded blastocysts were placed in the other drop, and 10-15 ES cells were injected per embryo.
All microscopic operations were performed under an inverted microscope. Manipulated embryos were transplanted into the uterine horns of ICR recipient females on the second day of pseudopregnancy after culturing for 1-2 hours. Recipient females who did not deliver pups even on the scheduled delivery date were subjected to caesarean section and fostered by foster parents. On day 4 of natural mating, ES cells 2B7 were injected into 160 blastocysts of C57BL / 6J mice collected by perfusing the uterus. As a result, 123 cells survived and the success rate was 77%. As a result of transplanting 123 into the uterine horns of ICR recipient females on the second day of pseudo-pregnancy, 103 were found to be implanted and 95 offspring were obtained. Of the 83 pups that reached weaning, 30 were able to be determined to be chimeric mice by coat color, and 26 of them were male in shape. The contribution rate of ES cells in these chimeric mice ranged from 10% to 95%, the contribution rate was less than 60% in 10 cases, 60% to less than 90% in 14 cases, and 90% or more in 2 cases. .
(B) The resulting chimeric mouse was mated with a C57BL / 6J mouse and tested whether the delivered offspring (F1 heterozygous mouse) was derived from a GLAST (GluT-1) gene-deficient ES cell. . If the germ cells of the chimeric mouse are derived from ES cells, the color of the baby born is wild, and if it is derived from the blastocyst of the C57BL / 6J mouse, it is black. Among the 9 chimeric mice (No. 1, 5, 13, 20, 33, 41, 54, 62, 81) with high ES cell contribution rate (over 70%), 8 cases (No. 1) to date , 5, 20, 33, 41, 54, 62, 81), the transmission of ES cells to the germ line was confirmed.
No. In a cross between 5 and C57BL / 6J female mice, a total of 23 pups were obtained in 3 deliveries, of which 23 showed a wild coat color. No. Of the 15 pups obtained by crossing 33 and C57BL / 6J female mice, 12 showed a wild coat color. As a result of analysis by Southern blotting on 23 of these wild-colored mice, the deletion of the GLAST (GluT-1) gene was confirmed in 11 cases.
(5) Backcrossing with C57BL / 6J strain mice F1 heterozygous male mice in which the GLAST gene deficiency was confirmed were mated with C57BL / 6J strain wildtype female mice to obtain offspring. The genotype was analyzed by Southern blotting, and next-generation heterozygous male mice in which the deletion of the GLAST gene was confirmed were selected. Again, this heterozygous male mouse was crossed with a C57BL / 6J female mouse to obtain the next generation heterozygous male mouse. In this way, the same mating was repeated 9 times in succession to obtain males and females of GLAST gene-deficient heterozygous mice with updated generations. Finally, by mating the male and female of this heterozygous mouse, a homozygous GLAST knockout mouse (GLAST − / −) for GLAST gene deficiency, a heterozygous GLAST knockout mouse (GLAST +/−) for GLAST gene deficiency, and Wild type mice (GLAST + / +) were obtained. These mice were used for the following measurements.
Example 2: Measurement of intraocular pressure In the homozygous GLAST knockout mouse and wild type normal mouse obtained in Example 1, intraocular pressure was measured at the time of 1 year after birth. After 4 animals in each group were sedated by anesthesia, the intraocular pressure was measured with an electronic tonometer. The results are shown below.
Wild type normal mouse: 19 ± 4 mmHg
Homo knockout mice: 15 ± 3 mmHg (mean ± standard deviation)
From this result, it was confirmed that the intraocular pressure of the homozygous GLAST knockout mouse was in the normal range.
Example 3: Measurement of the number of retinal ganglion cells In various ages, in the homozygous GLAST knockout mouse, heterozygous GLAST knockout mouse, and wild type normal mouse obtained in Example 1, hematoxylin / The number of retinal ganglion cells in each mouse was determined by eosin staining and retrograde labeling of retinal neurons with Fluoro-Gold or DiI.
Observation by section staining:
1) Test mice were sedated with a 1: 1 mixture of ketamine (10 mg / ml) / medetomidine (1 mg / ml) (0.15-0.2 ml / mouse) and perfusion-fixed with 4% paraformaldehyde solution. ,
2) Remove the eyeball and fix it in the same solution at 4 ° C for another 2 hours.
3) After embedding the eyeball in paraffin, a section, for example, a section having a thickness of 7 μm, was prepared. 4) Hematoxylin / eosin staining was performed, and nerve cells in the retinal ganglion region were observed under a microscope. This microscope image is shown in FIG.
5) The number of nerve cells in the section including the retinal ganglion part was counted for each mouse. The result is shown in FIG.
Observation by retrograde labeling:
1) After sedation with a 1: 1 mixture of ketamine (10 mg / ml) / medetomidine (1 mg / ml) (0.15-0.2 ml / mouse), the head of the mouse was fixed,
2) After spraying with ethanol under a microscope, the head is transected with scissors to expose the skull,
3) After confirming the suture line and blood vessel, open a hole for surgery with a grinder, and then inject Fluoro-Gold or DiI into the upper hill parenchyma with a microsyringe
4) After binding the skin with a clip, atipamezole (5 mg / ml) (0.15-0.2 ml / mouse) was injected intraperitoneally to confirm recovery.
5) After normal operation for 7 days after the operation, the treated mouse was killed by ether anesthesia, the eyeball was removed, and the anterior eye segment was removed.
6) Put the posterior eye part including the retina in 4% paraformaldehyde solution and fix at 4 ° C for 20 minutes,
7) The retina was taken out and a progress specimen was prepared, and 8) images of fluorescently labeled retinal ganglion cells were photographed with a fluorescence microscope. This image is shown in FIG.
From these results (FIGS. 3 to 5), it was found that in homozygous GLAST knockout mice, the number of neurons in the retinal ganglion was significantly reduced compared to wild type and heterozygous knockout mice. . As a result of hematoxylin / eosin staining (FIG. 3), the number of retinal ganglion cells indicated by arrows is decreased. In addition, a decrease in the number of cells (amacrine cells, bipolar cells, etc.) in the inner granule layer located in the center and accompanying thinning of the inner granule layer were observed. In homozygous GLAST knockout mice, the number of cells decreased from the time of birth, but in heterozygous knockout mice, the number of cells decreased with time, and after 5 weeks of age, the number of cells decreased significantly. It turned out that it has fallen to (FIG. 4).

  The homozygous or heterozygous GLAST knockout mouse deficient in the endogenous GLAST gene as a model mouse for normal-tension glaucoma according to the present invention has a therapeutic method for It is expected to be extremely useful for establishment and also for elucidating the pathophysiology of the disease, such as the onset cause, onset mechanism or progression mechanism.

Claims (5)

A method for producing a GLAST knockout mouse in which the function of the endogenous GLAST gene is deficient, wherein the GLAST knockout mouse is a GLAST knockout mouse in which the function of the endogenous GLAST gene is deficient, and the genetic background thereof is GLAST gene Except for 99% or more of the genetic background of C57BL / 6 mice,
1) the intraocular pressure is below 21 mmHg, and 2) the retinal ganglion cell count is reduced by at least 20% compared to C57BL / 6 wild-type mice,
A production method comprising the following steps 1 to 6:
1) obtaining any mouse ES cell that lacks the function of one endogenous GLAST gene on a homologous chromosome;
2) Obtaining a chimeric mouse comprising the ES cell obtained in the step 1,
3) crossing the chimeric mouse obtained in step 2 with a normal C57BL / 6 mouse to obtain a heterozygous knockout mouse;
4) crossing the heterozygous knockout mouse obtained in step 3 with a normal C57BL / 6 strain mouse to obtain a heterozygous knockout mouse;
5) Repeat the mating described in Step 4 at least nine times in total to obtain a heterozygous knockout mouse whose genetic background is close to that of the C57BL / 6 mouse, and 6) Heterozygous obtained in Step 5 Mating type knockout mice to obtain homozygous or heterozygous GLAST knockout mice. A homozygous or heterozygous GLAST knockout mouse produced using the production method of claim 1 and lacking the function of the endogenous GLAST gene , the genetic background of which excludes the GLAST gene , 99% or more identical to the genetic background of C57BL / 6 mice,
1) The intraocular pressure is 21 mmHg or less, and
2) The number of cells in the retinal ganglion is reduced by at least 20% compared to C57BL / 6 wild type mice,
GLAST knockout mouse . A method for screening a compound useful for the prevention and / or treatment of normal-tension glaucoma using the GLAST knockout mouse according to claim 2 . A method for screening a compound useful for the prevention and / or treatment of normal-tension glaucoma,
1) administering a test compound to the GLAST knockout mouse according to claim 2 ;
2) administering a test compound to a wild-type mouse,
3) In each of the above mice, evaluate the quantity or function of optic nerve cells that survive before and after a certain period of time, and 4) compare the test results of GLAST knockout mice and wild type mice. A screening method comprising evaluating the effectiveness of a test compound. The screening method according to claim 4 , wherein the number of neurons in the retinal ganglion is counted and / or the retinal potential is measured in order to evaluate the number of viable optic nerve cells or optic nerve cell function.

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